The Diagnostic Value Of MicroRNA-1290 In Colorectal Cancer,and The Role And Mechanism Of MicroRNA-1290 In The Formation Of Immunosuppressive Microenvironment

Background:Colorectal cancer（CRC）is one of the common gastrointestinal malignant tumors that seriously threaten human health,with high morbidity and mortality.Patient prognosis is closely related to the tumor stage.Therefore,finding a new non-invasive,convenient,and accurate tumor biomarker has become an urgent clinical problem that needs to be resolved.Liquid biopsy is of great value for finding effective biomarkers and establishing non-invasive detection methods.Among them,circulating miRNA has high stability,and can be used as a diagnostic marker for various diseases.Tumor cells and the tumor microenvironment（TME）are an interacting whole,and miRNAs play a key role as important post-transcriptional regulatory factors.Hypoxia,high concentration of lactic acid,lack of nutrients,etc.are important features of the TME,which play an important role in the occurrence and development of CRC.The TME not only recruits large number of immunosuppressive cells,such as Treg cells,MDSCs and TAMs,but also reshapes the infiltrated immune cells through metabolic reprogramming,impairs their immune surveillance and function,and promotes the formation of immune microenvironment.Among them,tumor cell-derived miRNAs participate in the formation of the immunosuppressive microenvironment through a variety of ways.Objective:The aim of the study is to screen effective diagnostic markers of CRC and to verify with clinical samples;on this basis,to explore the mutual regulation with the TME,especially the immunosuppressive microenvironment.Methods:The differentially expressed miRNAs of CRC patients were screened by serum miRNA sequencing,and the target gene prediction and functional enrichment analysis were performed to explore their possible biological function and molecular mechanism.Tissue expression level of differentially expressed miRNAs was analyzed through the TCGA database,and the serum expression level was verified through clinical samples.In order to evaluate the diagnostic value of miR-1290 in CRC,the serum miR-1290expression level of CRC,pancreatic cancer,gastric cancer,and healthy controls was detected by RT-q PCR,and the correlation analysis was carried out based on the clinical characteristics of patients.Finally,the role of miR-1290 in disease monitoring was analyzed by comparing the preoperative and postoperative serum miR-1290 levels,combined with the nude mouse subcutaneous xenograft model.In order to access the source of the high expression of miR-1290 in serum,RT-q PCR was used to analyzed the expression level of miR-1290 in CRC tissues,cell lines and cell supernatant.The effects of TME-related factors on the miR-1290 expression level,including hypoxia,glucose deprivation,lactic acid,ROS,TGF-β,IFN-γ,and senescence were analyzed by simulating the TME in vitro.The effect of miR-1290 on CRC progression was explored through in vitro and in vivo experiments.The role of miR-1290 in the metabolic adaptation of CRC was evaluated by detecting cellular glucose uptake,lactate secretion and ATP production.Using TCGA data for immune infiltration analysis,combined with in vitro experiments and animal models to analyze the effect of miR-1290 on the immune microenvironment.Result:1.Screening and identification of differentially expressed miRNAs in serum of patients with CRC.Through miRNA sequencing and differential expression analysis,94 up-regulated miRNAs were found in CRC serum;16 differentially expressed miRNAs were screened according to the fold change;target gene prediction and functional enrichment analysis suggested that differentially expressed miRNAs were involved in tumor progression.Combined with bioinformatics analysis and literature analysis,miR-203a-3p,miR-194-5p,miR-146-5p,miR-1290 and miR-96-5p were identified as candidate markers.The verification of clinical samples found that only miR-1290 was significantly up-regulated in serum of CRC（P<0.001）.2.Serum miR-1290 is a marker for diagnosis and disease monitoring of CRC.Though the detection and analysis of clinical samples,we found that the expression level of serum miR-1290 was significantly increased in CRC（P<0.05）,pancreatic cancer（P<0.01）,and gastric cancer（P<0.01）.In the diagnosis of CRC,the AUC was 0.7852（95%CI:0.6978-0.8726,p<0.0001）,the cut-off value was 11.79,the sensitivity was 42.00%,and the specificity was 90.00%;the combination with CEA improved the diagnostic efficiency.Animal models found that serum miR-1290 levels increased significantly after tumorigenesis（p<0.001）;after chemotherapy treatment,tumor growth was significantly inhibited and serum miR-1290 levels decreased（p<0.001）.What’s more,the postoperative serum miR-1290 expression level was significantly lower than that before the operation（p<0.05）.3.miR-1290 is a tumor-promoting molecule regulated by the TME.The expression level of miR-1290 was significantly increased in CRC tissues（p<0.05）;CRC cells highly expressed miR-1290,and secreted it into the cell supernatant.Hypoxia,glucose deprivation,ROS,TGF-β,IFN-γ,lactic acid and chemotherapeutic drugs-induced senescence in the TME could affect the expression of miR-1290 in CRC cells;In vitro and in vivo experiments showed that miR-1290promoted CRC tumor progression.4.miR-1290 affects the energy metabolism of CRC cells and participates in the formation of immunosuppressive microenvironment.miR-1290 promoted the glucose uptake and ATP production of CRC cells,and affectd the secretion of lactic acid.Immune infiltration analysis revealed that the expression level of miR-1290 was positively correlated with Th2 cell infiltration（r=0.660,p<0.001）,and negatively correlated with CD56^brightNK cell infiltration（r=-0.480,p<0.05）.Overexpression of miR-1290 up-regulated the expression of PD-1,TIGIT and TIM-3 in CD8⁺T cells,inhibited IFN-γsecretion,and promoted CD8⁺T cell exhaustion;overexpression of miR-1290 also induced infiltration of CD4⁺CD25⁺Treg cells and exerted immunosuppressive effects.Exosomes derived from CRC cells were highly enriched in miR-1290,suggesting that exosomes might participate in the formation of miR-1290-mediated immunosuppressive microenvironment.In conclusionSerum miR-1290 is a potential diagnostic biomarker in CRC.miR-1290 is highly expressed in CRC and its expression level is regulated by the TME.miR-1290participates in the metabolic reprogramming of CRC cells and promotes the formation of the immunosuppressive microenvironment.